To improve immunostaining of flat-mount retina the removal of the vitrous body is needed. It is, however, not a very easy task to perform.
One thing you can do if having problems with remaining vitreous, is to fix the eyeball for at least 4h in PBS with 4% paraformaldehyde at 4ºC right after enucleation. After which, you remove the anterior segment and lens, and once you isolate the retina you place 4 radial cuts in the periphery in order to keep it flatten. Then, I usually wash it with PBS containing 0.5-0.25% of triton X-100, which it seems to me that not only it helps to permeabilize the tissue, but also to remove part of the remaining vitreous that might left (I usually do this step adding the serum of the specie were the 2ari antibody was made), for ON at 4ºC with gentle shaking. I hope this can help: good luck!
I agree with you. A good tip is to use old animals, the vitreous (like in humans) is less adherent to the retina. In our lab we are cutting first the anterior segment (removal of the cornea with scissors at the limbus). Then you pull out the lens and most often you remove the vitrous body at the same time because it is adherent to the posterior capsule of the lens. Then do your 4 radial incisions through the sclera and the retina. Then gently remove the retina from the underlying RPE and sclera and cut it at the optic nerve head.
Good luck.
Alain Bron, University Hospital Dijon, France
One thing you can do if having problems with remaining vitreous, is to fix the eyeball for at least 4h in PBS with 4% paraformaldehyde at 4ºC right after enucleation. After which, you remove the anterior segment and lens, and once you isolate the retina you place 4 radial cuts in the periphery in order to keep it flatten. Then, I usually wash it with PBS containing 0.5-0.25% of triton X-100, which it seems to me that not only it helps to permeabilize the tissue, but also to remove part of the remaining vitreous that might left (I usually do this step adding the serum of the specie were the 2ari antibody was made), for ON at 4ºC with gentle shaking. I hope this can help: good luck!
Try intravitreal injection of homologous (mouse) plasmin (start with 1I.U./ml) before you fix your tissue, ideally in live animals. That should both liquefy and cleave Vitreoretinal adhesion.
I usually dissect the eye starting from the optic nerve and cutting it in half (going through the cornea) with a pair of microdissection scissors. After you do that, you can pull each piece away with forceps and the lens & vitreous separate from the rest of the eye. Alternatively, as people have already said, cut the anterior segment out and pull out the lens.
Good luck!
I was using piece of millipore filter paper on baboon retina. Just rub gently over the retina. Vitreous sticks to it.
I used to fix the retina AFTER removal of the vitreous body. I cut the anterior segment, then made three radial cuts and then took out the vireous body; some vitreous remains attache do the retina, so I removed it mechanically with watchmaker's forceps... if you have to do electrophysiology, or immunohistochemistry, some vitreous can give problems, so I uses collagenase to dissolve it. Then I flattened the retina with the ganglion cell layer up on a slide, and then put some drops of fixative on it, and let the retina to fix for some hours at 4°C in a closed capsule, with some fixative around to prevent drying of the retina.
Dear Alessandro
Thanks for your suggestions. I use to remove the retina before fixation in the rat which by some reason is much easier than in mouse where moch more of the vitreous sticks to the retina. What concentrations are you using of collagenase and for how long time?
best wishes
Jens
With adult mouse retinas I've found that taking out the lens pulls most of the vitreous with it, the remaining I remove with soft brushes after flattening out the retina with relieving cuts.
My usual protocol for immunohistochemistry is after enucleation put the eye into 4% PFA, remove the anterior segment including the lens while in PFA (I've found this short fix helps prevent pulling out the retina along with the lens). Leave the eyecup in fix for the desired amount of time (between 10-30mins). Then dissect out the retina after rinsing with PB.
Another method I've seen used for removing vitreous (but in the primate) is the "fire-hose" method where a stream of PB is pumped through a syringe to "blow-away" the vitreous. This method also requires mounting on filter paper first so the retina has a bit more support.
The strongest points of attachment of the vitreous are around the optic nerve head, fovea and at the anterior ciliary/iridial attachments. If you use very fine forceps to break these attachments and then use same fine toothed forceps to pinch just anterior to the retina., ie to grasp the vitreous gel, you should be able to remove the vitreous in one piece. I would not fix in 4% paraformaldehyde before you remove the vitreous.
I have attached a pdf of a paper where I have detailed the technique for retinal wholemount preparation which may be helpful.
Dear Tailoi chan-ling
Thanks for your paper. I will try to transfer your technique to the mouse retina.
Best wishes
Jens
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